Fabry disease is an X-linked inborn error of glycolipid metabolism caused by a deficiency of the lysosomal enzyme, alpha-galactosidase A. In affected males this leads to early death due to occlusive disease of the heart, kidney, and brain. The lack of sufficient quantities of purified enzyme has prevented a complete evaluation of the potential efficacy of enzyme replacement therapy in Fabry disease. In order to obtain large quantities of this enzyme, we previously constructed baculovirus derivatives that produced relatively low levels (2-5 mg/liter) of the human enzyme. In this proposal we wish to determine if production of the recombinant enzyme can be increased to much higher levels using Pichia pastoris. This yeast host system has been used to produce a variety of heterologous proteins at levels that range up to several grams per liter. P. pastoris carries out eucaryotic type post-translational processing, including glycosylation. We propose to (i) clone the cDNA for the human alpha-galactosidase A to plasmid pPICZ-alpha A that has the yeast alpha- factor signal peptide so that the human enzyme will be secreted, (ii) adapt the current purification scheme to the PerSeptive Biosystems BioCAD HPLC Workstation of the type used in GMP production, and (iii) determine the composition and sequence of the carbohydrate on the human alpha- galactosidase A since the type of carbohydrate present on glycoproteins can have important effects on biological activity, clearance in the serum, and cellular uptake.
An estimated 1,500 patients (minimal estimate) in the United States would be expected to pay perhaps $250,000 per year, every year, from onset of symptoms (approximately age 20) for approximately 40 years. (1,500 X $250,000 = $375,000,000 per year].
| Chen, Y; Jin, M; Egborge, T et al. (2000) Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris. Protein Expr Purif 20:472-84 |
